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Biomedical subjects

J E Thomas

Publications and source records attributed to J E Thomas.

At least 73 records · Page 4Linked to original sources
PubMed1997

Inhibition of MG-63 cell proliferation and PDGF-stimulated cellular processes by inhibitors of phosphatidylinositol 3-kinase.

Studies on a platelet-derived growth factor (PDGF) responsive osteosarcoma cell line, MG-63, were initiated to determine the effects of phosphatidylinositol (Ptdlns) 3-kinase inhibitors on serum-stimulated cell proliferation and PDGF-stimulated DNA replication, actin rearrangements, or Ptdlns 3-kinase activity. In a dose-dependent manner, the fungal metabolite wortmannin and a quercetin derivative, LY294002 (2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one), inhibited serum-stimulated MG-63 cell proliferation. The mitogenic effects of PDGF on MG-63 cells, as determined by incorporation of [3H]-thymidine, were also substantially inhibited in the presence of 0.10 microM wortmannin or 10 microM Ly294002. Furthermore, MG-63 cells stimulated by PDGF form distinct actin-rich, finger-like membrane projections which are completely inhibited by either 0.10 microM wortmannin or 10 microM LY294002. At these same concentrations, wortmannin and LY294002 were also effective at reducing levels of phosphatidylinositol 3-phosphate in PDGF-stimulated MG-63 cells. Treatment of these cells with increasing concentrations of wortmannin reduced the level of PDGF stimulated tyrosine phosphorylation of the PDGF receptor but did not significantly affect the amount of the Ptdlns 3-kinase regulatory subunit, p85, associated with the receptor. Additionally, pretreatment of cells with 0.250 microM wortmannin followed by stimulation with PDGF resulted in a slightly reduced level of receptor autokinase activity; however, similar treatment with 50 microM LY294002 did not affect the level of autokinase activity. These results demonstrate the effects of two different Ptdlns 3-kinase inhibitors on serum- and PDGF-stimulated MG-63 cell proliferation and PDGF-stimulated morphological changes and suggest a greater role for Ptdlns 3-kinase in these processes.

Androstadienes↗
PubMed1997

Carbon 13-labeled urea breath test for the diagnosis of Helicobacter pylori infection in children.

Helicobacter pylori infection is mainly acquired in childhood, and studies on the epidemiology of this infection depend on the availability of a noninvasive diagnostic test for use in children. The aim of this study was to determine whether the carbon 13-labeled urea breath test (UBT) can be used in children by evaluating: (1) its sensitivity and specificity compared with either culture or both rapid urease test and histologic examination, (2) whether a test meal or a prolonged fast is required, (3) the usefulness after treatment for H. pylori. Eighty-eight children (mean age, 10.6 +/- 4.19 years) who were undergoing upper endoscopy were studied while fasting, not fasting, and after treatment. Children were given 50 mg of 13C-urea if they weighed less than 50 kg or 75 mg of 13C-urea if they weighed more than 50 kg with 50 mg of a glucose polymer solution in 7.5 ml of water. Breath samples were collected at baseline and at 15, 30, 45, and 60 minutes. In 63 fasting children the UBT was 100% sensitive and 97.6% specific at 30 minutes with a cutoff value of 3.5 delta 13CO2 per mil. Nonfasting tests in 23 children, performed between 1 and 2 hours after their usual meal, were 100% sensitive and 91.6% specific. In 13 children fed directly before the UBT, the sensitivity of the test was reduced to 50%. Thirty minutes was the optimal sampling time. There was a significant decrease in specificity when samples were obtained at 15 minutes, possibly caused by the interference of oral urease-producing organisms. The test was 100% sensitive and specific in 20 children after treatment for H. pylori infection. The UBT is a highly sensitive and specific test for the diagnosis of H. pylori infection in children. Neither a prolonged fast nor a test meal is required.

Adolescent↗
PubMed1997

Rapid reversal of endothelin-1-induced cerebral vasoconstriction by intrathecal administration of nitric oxide donors.

OBJECTIVE: To determine the capability of donors of nitric oxide (NO) (sodium nitroprusside, nitroglycerine) to reverse endothelin-1 (ET-1)-induced cerebral vasoconstriction in vivo, when administered through the cerebrospinal fluid (CSF) to the adventitial side of the constricted blood vessel. METHODS: The rabbit basilar artery was exposed through a transcervical, transclival approach and subsequently subjected to pharmacological manipulations and direct observation of effects by videomicroscopy. Specific manipulations were suffusion of ET-1 (100 nmol/L, 1 ml/min) in synthetic CSF (sCSF) to provoke vasoconstriction and then either suffusion of an NO donor in sCSF (2 mg/ml/min), or sCSF alone. The second suffusion was always made separately and begun during the period of stable maximal vasoconstriction, which occurred between 20 and 30 minutes after beginning the first suffusion. Measurements of the diameter of the artery were made using an inline video caliper. RESULTS: Sodium nitroprusside and nitroglycerine, both donors of NO, rapidly and completely reversed ET-1-induced vasoconstriction without causing hypotension. The average value for maximal vasoconstriction by ET-1/sCSF was 50.4% of baseline arterial diameter and occurred between 20 and 30 minutes. The rate of vasodilatory response was 100% of significantly constricted arteries. The response was complete in less than 6 minutes in all preparations, as compared to the 60 minutes required for spontaneous relaxation (sCSF suffusion alone). CONCLUSION: NO donors are effective in reversing cerebral vasoconstriction when administered intrathecally, cause no significant hemodynamic change when so administered, and may represent an important therapeutic intervention for cerebral vasospasm.

Animals↗
PubMed1997

Inter- and intraspecies comparison of the 16S-23S rRNA operon intergenic spacer regions of six Listeria spp.

The 16S-23S rRNA intergenic spacer (IGS) regions found in six Listeria species were characterized. PCR amplification of the 16S-23S IGS with a "generic primer" set generated products of about 340 bp (small) and 550 to 590 bp (large) with DNA from all Listeria strains tested. Seven Listeria monocytogenes serotype 4b strains and one L. monocytogenes serotype 4d strain also had an additional PCR product of ca. 360 bp. The 360-bp PCR product from one of these L. monocytogenes serotype 4b strains was identical in nucleotide sequence to the small 340-bp IGS, except that it contained an 18-bp tandem repeat. The small rRNA IGSs of L. innocua, L. ivanovii, L. seeligeri, L. welshimeri, and L. grayi were 83 to 99% homologous to that of L. monocytogenes. The large rRNA IGS of L. monocytogenes was 81 to 96% homologous to those of the other Listeria species and agreed with current taxonomic division among these species. The nucleotide sequences of the central 274 bp of the large rRNA IGS of strains from seven different L. monocytogenes serotypes were highly homologous; however, serotype-specific differences were noted, and four groups were identified within L. monocytogenes based on this analysis.

Animals↗
PubMed1997

Induction of phosphorylation on BRCA1 during the cell cycle and after DNA damage.

BRCA1, the familial breast cancer susceptibility gene product, is a 220-kDA phosphorylated protein. BRCA1 immunoprecipitated from MCF7 cells blocked in G1-S phase or progressing through S-phase of the cell cycle migrated more slowly through SDS polyacrylamide gels than BRCA1 from cells maintained in serum-supplemented media, serum-free media for 24 h, or delayed in G2-M phase by treatment with colchicine. Restoration of BRCA1 to the faster-migrating form, which occurred on release of cells from the G1-S-phase block, was prevented by the phosphatase inhibitor okadaic acid. Phosphatase treatment of immunoprecipitated BRCA1 resulted in the conversion of the slower-migrating form to the faster-migrating form. Although these results suggested that BRCA1 was preferentially hyperphosphorylated near the G1-S-phase boundary of the cell cycle, exposure of cells to DNA-damaging agents including UV light or treatment with hydrogen peroxide (H2O2) also promoted BRCA1 hyperphosphorylation. These same stimuli also eliminated the punctate nuclear staining pattern normally observed for BRCA1 in control cells. These results indicate that BRCA1 undergoes cyclic hyperphosphorylation during the cell cycle; however, this modification, as well as changes in BRCA1 nuclear staining, also occurs in response to DNA damage.

Aphidicolin↗
PubMed1996

Subcellular localization and analysis of apparent 180-kDa and 220-kDa proteins of the breast cancer susceptibility gene, BRCA1.

The breast cancer susceptibility gene BRCA1 encodes an 1863-amino acid protein that acts as a tumor suppressor. The biochemical function of BRCA1 is unknown, and there are conflicting results describing its subcellular location. We have identified a 220-kDa protein, which is reactive with three antibodies raised against the amino- and carboxyl-terminal regions of BRCA1. Immunoflourescence staining with an antibody to the carboxyl terminus of BRCA1 localized the protein to the nucleus of breast, ovarian, and cervical carcinoma-derived cell lines. A similar result was observed by biochemical subcellular fractionation that indicated that the 220-kDa protein was localized primarily to the nucleus of cell lines established from breast carcinomas. In addition to the 220-kDa protein, one antibody, C-20, also recognized a 180-kDa protein in MDA-MB-468 total cell lysates that was not detected by the other two antibodies. Several observations suggest the 180-kDa protein is the epidermal growth factor (EGF) receptor: (i) C-20 reacted avidly with a 180-kDa protein immunoprecipitated by an antibody to the EGF receptor; (ii) an EGF receptor antibody detected a 180-kDa protein immunoprecipitated by C-20; (iii) the affinity purified EGF receptor was both immunoprecipitated and detected on immunoblots by the C-20 antibody but not another BRCA1 antibody; (iv) similar phosphopeptide maps were generated from the EGF receptor and the 180-kDa protein immunoprecipitated by C-20, and this peptide map was distinct from the 220-kDa phosphoprotein; and (v) the C-20 immunizing peptide bears sequence identity to the EGF receptor. These results indicate that BRCA1 is a 220-kDa nuclear protein and that the 180-kDa protein reported previously may be unrelated to BRCA1.

Antibodies↗
PubMed1996

Development and characterization of a porcine model to study osteoclast differentiation and activity.

The study of osteoclast differentiation, function, and fate has been hampered by the lack of nonavian, nonrodent models in which biochemical and molecular studies can be conducted. The present study was undertaken to determine if osteoclasts could be generated from porcine bone marrow cells. Bone marrow from the long bones of neonatal female pigs was enriched for mononuclear cells and cultured in the presence or absence of 1,25-(OH)2D3, rhIL-11, or PGE2. A confluent layer of stromal cells was observed after 4-8 days in culture and multinucleated giant cells formed after 6-10 days of culture. The multinucleated cells stained positively for tartrate-resident acid phosphatase and formed resorption lacunae when exposed to bovine cortical bone slices. When examined by transmission electron microscopy, abundant mitochondria, perinuclear Golgi complexes, numerous variably sized vacuoles, prominent rough endoplasmic reticulum, and free polysomes were observed in the multinucleated cells. Stimulation of the in vitro generated osteoclasts with 10(-8) mol/L salmon calcitonin resulted in a three to fivefold increase in cAMP production and in cell retraction. Although the osteoclasts formed in the presence or absence of 1,25-(OH)2D3, 10-50-fold more osteoclasts were observed in the cultures treated with 1,25-(OH)2D3 in comparison to cultures without 1,25-(OH)2D3. Osteoclast differentiation was also stimulated by rhIL-11 and PGE2; although, the number of cells generated in 6-7 days was significantly less than the number obtained with 1,25-(OH)2D3, treatment. In addition, these multinucleated cells expressed high levels of Src kinase activity and responded to bafilomycin A1, an inhibitor of the vacuolar type H(+)-ATPase, treatment with a decrease in osteoclastic bone resorption. In summary, the porcine cells possess the major distinguishing characteristics of osteoclasts and provide an alternative mammalian model to study osteoclast differentiation and resorptive activity.

Animals↗
PubMed1996

Genus- and species-specific detection of Listeria monocytogenes using polymerase chain reaction assays targeting the 16S/23S intergenic spacer region of the rRNA operon.

In this study, the 16S/23S rRNA intergenic spacer (IGS) regions of six Listeria species were examined. DNA bands of 590 and 340 bp were observed following polymerase chain reaction (PCR) amplification of DNA from Listeria monocytogenes, Listeria innocua, Listeria seeligeri, Listeria welshimeri, and Listeria ivanovii strains with generic rRNA IGS oligonucleotide primers. For strains of Listeria grayi subspp. grayi and murrayi, DNA band sizes of 550 and 340 bp were observed with this primer pair. DNA bands of these sizes were not observed for other Gram-negative or- positive bacteria in this PCR assay. Four RsaI digestion profiles were noted for the Listeria PCR products. Listeria monocytogenes strains had one profile; L. innocua strains had a second; L seeligeri, L. welshimeri, and L ivanovii strains had a third; and L. grayi strains had a fourth. The small and large 16S/23S rRNA IGSs of L. monocytogenes ATCC 15313 were identical in the first 58 5' and the last 169 3' nucleotides. However, the large rRNA IGS contained a central 267-bp region with tRNA(Ile) and tRNA(Ala) genes. Large rRNA 16S/23S IGS nucleotide sequence data has not been previously reported. This data was used to develop novel Listeria genus-specific and L.monocytogenes species-specific PCR assays.

Base Sequence↗
PubMed1995

Three members of the nitric oxide synthase II gene family (NOS2A, NOS2B, and NOS2C) colocalize to human chromosome 17.

Nitric oxide synthases (NOSs) are a family of enzymes responsible for the synthesis of nitric oxide from L-arginine and molecular oxygen. Three human NOS enzymes (I, II, and III) with differing cellular distribution and regulatory mechanisms have been identified. To determine whether additional NOSs are encoded in the human genome, a bovine NOS II-related cDNA was used to screen two human genomic libraries. Clones containing three independent genes were isolated. One clone encoded the previously identified NOS II gene (NOS2A). The two other genes specified amino acids homologous, but not identical, to human NOS II (NOS2B and NOS2C). Southern blot hybridization demonstrated that all three genes are present in the human genome. DNA from human-mouse somatic cell hybrids were used to determine the chromosomal location of the NOS II-related genes. All three NOS II-related genes colocalized to human chromosome 17 between bands p13.1 and q25. These observations suggest that there is more than one NOS II-related gene in the human genome. This finding may have important implications for the design of NOS isoform-specific inhibitors.

Amino Acid Sequence↗
PubMed1995

The feisty family.

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Adenocarcinoma↗
PubMed1994

Cerebral imaging in the asymptomatic preoperative bronchogenic carcinoma patient: is it worthwhile?

The issue of screening for cerebral metastatic disease in the preoperative bronchogenic carcinoma patient remains unsettled and changes with advancing technology. A prospective nonrandomized study was designed to compare contrast magnetic resonance imaging (MRI) with computed tomography (CT) after several clinical situations suggested improved sensitivity for the former study. Patients with clinically operable disease and normal neurologic examinations were referred for both enhanced cerebral CT and MRI studies. Forty-two patients were entered and completed the enhanced CT scan; only 30 tolerated the MRI. The demographic data and histology of the patients appeared fairly typical for a series of operative candidates. No unsuspected metastatic lesion was found in this selected and low-risk group. We conclude that neither MRI nor enhanced CT scan is indicated in the asymptomatic bronchogenic carcinoma patient due to expense and lack of positive findings. Magnetic resonance imaging demonstrated more subtle benign pathology, but this study did not allow comparison of the two techniques in detection of metastatic disease.

Aged↗
PubMed1994

Detection of an unidentified potyvirus from Roystonea regia palm using the polymerase chain reaction and degenerate, potyvirus specific, primers and potential problems arising from the amplification of host plant DNA sequences.

Degenerate potyvirus-specific primers were used in the PCR to amplify cDNA representing a 335 nucleotide region of the coat protein gene in RNA purified from an uncharacterised potyvirus isolated from Roystonea regia palms in Queensland, Australia. The RNA was also detected by PCR in total nucleic acid extracts from infected Nicotiana benthamiana, N. clevelandii and Vicia faba. The method also successfully detected pea seed-borne mosaic virus in Pisum sativum. In addition the procedure amplified DNA's of approximately 200 bp and 420 bp, of non-viral origin, from total nucleic acid extracts of healthy Nicotiana spp, indicating that size of the PCR products needs to be included as a criterion for identifying virus specific products when this method is used.

Base Sequence↗
PubMed1994

Multiple sclerosis in black South Africans and Zimbabweans.

Multiple sclerosis is rare among the indigenous black people of Africa. The first account of a black patient with multiple sclerosis in South Africa was published as late as 1987. Since then a search to find black patients with multiple sclerosis in Southern Africa has continued. Seven black patients have now been traced in South Africa and five in Zimbabwe in whom a diagnosis of multiple sclerosis can be accepted. Six of the 12 patients became blind, or nearly so, from severe optic neuritis. Multiple sclerosis in these few black patients more often resembled the disorder as it occurs in oriental people than among white people in southern Africa or the black people of North America or the Caribbean.

Adolescent↗